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Goats distinguish between positive and negative emotion-linked vocalisations

Baciadonna, Luigi; Briefer, Elodie F.; Favaro, Livio; McElligott, Alan G.

Published in: Frontiers in Zoology

DOI: 10.1186/s12983-019-0323-z

Publication date: 2019

Document version Publisher's PDF, also known as Version of record

Document license: CC BY

Citation for published version (APA): Baciadonna, L., Briefer, E. F., Favaro, L., & McElligott, A. G. (2019). Goats distinguish between positive and negative emotion-linked vocalisations. Frontiers in Zoology, 16, [25]. https://doi.org/10.1186/s12983-019-0323-z

Download date: 28. sep.. 2021 Baciadonna et al. Frontiers in Zoology (2019) 16:25 https://doi.org/10.1186/s12983-019-0323-z

RESEARCH Open Access Goats distinguish between positive and negative emotion-linked vocalisations Luigi Baciadonna1*, Elodie F. Briefer2,5*, Livio Favaro3,6 and Alan G. McElligott1,4*

Abstract Background: Evidence from humans suggests that the expression of emotions can regulate social interactions and promote coordination within a group. Despite its evolutionary importance, social communication of emotions in non-human animals is still not well understood. Here, we combine behavioural and physiological measures, to determine if animals can distinguish between vocalisations linked to different emotional valences (positive and negative). Using a playback paradigm, goats were habituated to listen to a conspecific call associated with positive or negative valence ( phase) and were subsequently exposed to a variant of the same call type (contact call) associated with the opposite valence (dishabituation phase), followed by a final call randomly selected from the habituation phase as control (rehabituation phase). The effects of the calls on the occurrence of looking and cardiac responses in these phases were recorded and compared. Results: We found that when the valence of the call variant changed, goats were more likely to look at the source of the sound, indicating that they could distinguish calls based on their valence. Heart rate was not affected by the valence of the calls played, whereas heart-rate variability tended to be higher in the habituation and rehabituation phases, when positive calls were played compared to negative ones. Together, the behavioural and physiological measures provide evidence suggesting, first, that goats are able to distinguish call variants based on their valence, and second, that goat behaviour and cardiac responses are affected by call valence. Conclusion: This study indicates that auditory modalities are a potent means to communicate emotions in non- human animals. These findings can contribute to our understanding of the evolution of emotion perception in non-human animals. Keywords: Bioacoustics, Emotions, Heart-rate variability, Playback, Positive and negative valence, Ungulates

Background widespread, and their basic underlying mechanisms might Emotions have an adaptive value because they allow ani- be preserved across taxa [5]. mals to respond appropriately to salient stimuli. Negative In comparative , substantial progress has emotions enable individuals to respond appropriately to been made in identifying animal emotions using behav- potentially life-threatening situations. Positive emotions, ioural [6, 7], physiological [8], and cognitive indicators by contrast, guide responses to stimuli or events that [9, 10]. Accordingly, emotions are often accompanied by enhance fitness and widen the individual cognitive and be- visible changes in a subject’s facial expression, behaviour havioural repertoire [1–4]. Given the adaptive importance [11, 12] and vocalisations [6, 13, 14]. Although emotion- of emotions, their occurrence should be phylogenetically related changes are not necessarily intentionally commu- nicated, they could be used as cues to the emotional states of conspecifics [15, 16]. Behavioural and physio- * Correspondence: [email protected]; [email protected]; logical responses of the receivers of the cue can be used [email protected]; [email protected] to assess whether these animals simply perceive the 1Biological and , School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, difference between emotional stimuli or whether they UK are also affected by these stimuli in a way that matches 2 Institute of Agricultural Sciences, ETH Zürich, Universitätsstrasse 2, 8092 the emotion of the producer of the cues [17–21]. Zürich, Switzerland Full list of author information is available at the end of the article

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Baciadonna et al. Frontiers in Zoology (2019) 16:25 Page 2 of 11

Non-human animals are able to perceive the We assessed the effect of the perception of emotional- emotional state of conspecifics and even heterospecifics linked calls at a physiological level by recording heart (including humans) by using one sensory modality or a rate (HR) and heart-rate variability (HRV) during the combination of sensory modalities [22–24]. Additionally, playback experiments. HR is controlled by activation of state-matching of emotions between producers and the sympathetic (increase in HR) and vagal (decrease in receivers has been shown in some species [20, 25, 26]. HR) systems and therefore is considered an indicator of For example, exposure to odour or vocal cues from a emotional [7, 38]. By contrast, HRV is mainly stressed individual in cattle (Bos taurus) and pigs (Sus under the vagal regulation and thus indicates when only scrofa) or witnessing a family member being involved in the vagal branch of the autonomic nervous system is an agonistic interaction in geese (Anser anser) can affect activated [39, 40]. Because low HRV is associated, in the behaviour of the subject by increasing fearfulness humans, with depression and post-traumatic stress dis- and modifying physiology (cortisol level and heart rate; order, it has been proposed as a potential indicator of 25–27) [26–29]. Horses (Equus caballus) show a left emotional valence in other species [39–43]. Overall, gaze bias and increased heart rate when facing photos of physiological parameters can provide strong evidence in angry human faces compared to happy human faces addition to behavioural data when investigating the [30]. In rodents, exposure to negative ultrasonic calls arousal and valence of emotions [7, 44, 45]. We pre- causes anxiety-related behaviours, while exposure to dicted that HR would decrease during the habituation positive sounds triggers approach behaviours [31, 32]. phase and increase when the dishabituation stimulus Further evidence is required to understand the mechan- was played regardless of the valence of calls, while the ism through which emotions affect conspecifics [33]. opposite would occur for HRV. We hypothesized that Goats (Capra hircus) are highly social and are an excel- HRV, because it negatively correlates with HR, would lent model to investigate the mechanisms underlying the increase over time during the habituation phase. Fur- social dimension of emotions. Goat contact calls encode thermore, in the dishabituation phase, we expected an important information about the arousal and valence of increase in HR and therefore predicted reduced HRV the emotional state of the caller, along with information regardless of call valence upon hearing the first call. We on caller’s individuality, sex and age [6, 30]. Accordingly, it also expected an increase in HRV over the two subse- is likely that the expression of emotions in goat contact quent calls, as for the habituation phase. calls can be detected by other members of the group in a In this study, we considered several critical issues often similar way to other types of information [33–35]. In not controlled in related research: 1) the emotional state addition, goats are sensitive to human facial expressions of both the caller and the receiver were assessed, and 2) and show a preference for happy compared with angry only contact calls were used, so that the reaction of the faces [36]. The aims of this study were to investigate receiver would be purely dependent on the encoded emo- whether goats can discriminate conspecific calls conveying tions rather than the type of vocalisations [46]. positive and negative emotional information and to assess the potential impact of the emotional valence conveyed by Results the calls on the behavioural and physiological responses of Occurrence of looking towards the speaker receivers. In particular, to achieve our aims, we used a During the habituation phase (calls H1-H9), goats re- habituation-dishabituation-rehabituation playback design duced the occurrence of looking towards the speaker 2 [37], in which conspecific contact calls recorded during (Generalised Linear Mixed-Effect Model: χ (1) = 30.01, situations triggering emotions of positive or negative p < 0.0001; Fig. 1), indicating that they habituated to the valence were played back to the goats in the habituation call type, regardless of call valence (GLMM; valence: χ2 phase, before changing the valence in the dishabituation (1) = 0.13, p = 0.71; interaction between call number and 2 phase. We expected to find an increased level of attention valence: χ (1) = 0.26, p = 0.60). There was a tendency for (looking towards the source of the sound for longer) when goats to further reduce the occurrence of looking the dishabituation stimulus was played, if perceived as between the last call of habituation (H9) and the 1st call 2 different from the calls used in the habituation phase. In of dishabituation (D10; GLMM; χ (1) = 3.76, p = 0.052), addition, to verify that the response pattern in the dishabi- regardless of the valence of the calls (GLMM; valence: χ2 tuation phase was not due to a random change of atten- (1) = 0.18, p = 0.66; interaction between call number and 2 tion, the playback ended with the same variant of calls valence: χ (1) = 1.63, p = 0.20). When the last call of used during the habituation phase. In this control habituation (H9) and the 2nd call of dishabituation condition, we expected shorter duration of looking (D11) were compared, we did not find any significant ef- towards the source of the sound compared to the dishabi- fect of call number, valence or their interaction (p ≥ 0.18) tuation phase, because the calls would have been played . During the dishabituation phase, subjects increased the already during habituation. occurrence of looking between the 1st (D10) and the Baciadonna et al. Frontiers in Zoology (2019) 16:25 Page 3 of 11

Fig. 1 Occurrence of looking in response to the playbacks. The mean +/− SE occurrence of looking towards the loudspeaker is indicated in light grey for Positive (H1-H9)-Negative (D10-D12)-Positive (R13) call sequences in dark grey for the Negative (H1-H9)-Positive (D10-D12)-Negative (R13) call sequences. *** p < 0.001; ** p < 0.01; * p < 0.05

2 2nd call (D11; GLMM; χ (1) = 5.58, p = 0.018), regardless change in call valence, indicated by a drop in the occur- 2 of the valence of the calls (GLMM; valence: χ (1) = rence of looking suggesting a freezing response. 0.004, p = 0.94; interaction between call number and 2 valence: χ (1) = 0.88, p = 0.34). Call number, valence or Physiology: heart rate (HR) and heart rate variability their interaction (p ≥ 0.53) were not significant when the (HRV) 2nd (D11) and 3rd (D12) calls were compared. Finally, HR decreased during habituation (calls H1-H9; LMM; χ2 the occurrence of looking decreased between the 2nd (1) = 26.24, p < 0.001; Fig. 2), but did not change during call of dishabituation (D11) and the rehabituation call the dishabituation or rehabituation phases. Neither the 2 (R13; G LMM; χ (1) = 8.12, p = 0.004). Additionally, they valence of the calls played during the habituation phase 2 looked more at the call (D11 or R13) that was negative (LMM; χ (1) = 2.50, p = 0.11) nor the interaction be- 2 (frustration and isolation calls combined; mean ± SD = tween call number (H1-H9) and valence (LMM; χ (1) = 0.41 ± 0.14) compared to the positive call (0.21 ± 0.11; G 0.31, p = 0.57) had an effect on HR. When the last habit- 2 LMM; χ (1) = 8.12, p = 0.004). The interaction between uations call (H9) and the dishabituation calls (D10, D11, call number and valence was not significant (D11 vs and D12) were analysed, HR was not affected by call 2 R13; χ (1) = 0.00, p = 1.00). Call number, valence or their number, valence or their interaction (p ≥ 0.97). When interaction on the occurrence of looking between the 1st the dishabituation calls (D10 vs D11 and 11 vs 12) were dishabituation call (D10) and the rehabituation call (R13; considered, HR was not affected by call number, valence p ≥ 0.40) were not significant. When the 3rd call of dish- or their interaction (p ≥ 0.09). Finally, when the calls of abituation (D12) and the rehabituation call (R13) were dishabituation (D10, D11, and D12) and the rehabitua- analysed, we found that goats looked more at the call tion call (R13) were considered, HR was not affected by (D12 or R13) that was negative (mean ± SD = 0.29 ± 0.13) call number, valence or their interaction (p ≥ 0.23). compared to the positive call (0.17 ± 0.10;GLMM; χ2 There was a possible interaction effect between valence 2 (1) = 5.38, p = 0.020). However, call number (χ (1) = 1.68, and call number for heart-rate variability during habitu- 2 p = 0.19) and the interaction between call number (D12, ation (calls H1-H9; LMM; χ (1) =3.75, p =0.052;mean± 2 R13) and valence (χ (1) = 0.20, p = 0.65) were not signifi- SD positive valence = 57.16 ± 1.87 ms vs negative valence = cant. In summary, when the valence changed from the last 53.55 ± 2.39 ms; Fig. 3). However, post-hoc Tukey tests call of habituation to the first call of dishabituation, goats investigating the valence effect on each habituation call tended to decrease the occurrence of looking. This behav- did not reveal any statistical differences in HRV between iour then increased to reach a similar value as at the end positive and negative calls (p > 0.74). The comparison of the habituation when the second call of the dishabitua- between the last call of habituation (H9) and the 1st call tion was played back. This suggests an ability to detect a of dishabituation (D10) revealed that HRV was higher for Baciadonna et al. Frontiers in Zoology (2019) 16:25 Page 4 of 11

Fig. 2 Heart rate during the playbacks. Mean +/− SE heart rate (HR) during the habituation phase (H1-H9), dishabituation phase (D10-D12) and rehabituation phase (R13). During the habituation phase, HR decreased and did not vary significantly throughout dishabituation and rehabituation. *** p < 0.001; NS = not significant the call (H9 or D10) that was positive (mean ± SD = 51.83 ± 7.20 ms; z =2.44,p = 0.067). All the other compari- 59.59 ± 4.95 ms) compared to the negative call (48.53 ± sons included in the post-hoc analyses were not significant 2 6.1 ms; LMM; χ (1) =4.37, p = 0.036), regardless of call (p ≥ 0.17). In summary, HR decreased during habituation 2 number (LMM; χ (1) =0.03, p = 0.86; interaction between and did not change in the dishabituation and rehabitua- 2 call number and valence LMM; χ (1) =1.58,p =0.20).The tion phases. Heart-rate variability tended to be higher comparison between the 1st (D10) and 2nd (D11), and be- when positive calls were played compared to negative ones tween the 2nd (D11) and 3rd (D12) calls of dishabituation in the habituation and rehabituation phases. did not reveal any significant effect of call number, valence or their interaction (p ≥ 0.61). Finally, the 3rd call (D12) of Discussion dishabituation was compared to the rehabituation call The ability of goats to discriminate the emotional (R13) and an interaction effect was found between call valence conveyed by conspecific calls and the effect of 2 number and valence (LMM; χ (1) =4.36, p = 0.036). Post- these calls on their physiology were investigated using a hoc analyses revealed a tendency for the HRV to be lower habituation-dishabituation-rehabituation paradigm. We for negative rehabituation calls (R13; mean ± SD = 51.76 ± provide evidence which suggests that goats are probably 7.33 ms) than for positive rehabituation calls (70.12 ± 3.52 able to discriminate between conspecific calls associated ms; Tukey HSD; z =2.45, p = 0.064). There was also a with opposing emotional valences. Behavioural and tendency for the HRV to be higher when the rehabituation physiological responses suggest that the discrimination call was positive (R13; mean ± SD = 70.12 ± 3.52 ms) than occurred with a short delay and on the second call of when the 3rd dishabituation call was positive (D12; dishabituation. Our study suggests that vocalisations are

Fig. 3 Heart-rate variability (RMSSD) in response to the playbacks. Mean +/− SE RMSSD during the habituation phase (H1-H9), dishabituation phase (D10-D12) and rehabituation phase (R13). The black line (PNP) represents the sequence positive (habituation) – negative (dishabituation) – positive (rehabituation) calls and the grey line (NPN) represents the sequence negative (habituation) – positive (dishabituation) – negative (rehabituation) calls. The habituation phase revealed an interaction effect between the valence of the call broadcasted and the call number (H1- H9). The comparison between the last call of habituation (H9) and the 1st call of dishabituation (D10) revealed an effect of valence. An interaction effect between call number and valence was found when the 3rd call of dishabituation (D12) was compared with the rehabituation call (R13). *** p < 0.001; ** p < 0.01; * p = 0.052; NS = not significant Baciadonna et al. Frontiers in Zoology (2019) 16:25 Page 5 of 11

a potentially powerful channel for expressing emotions the calls before reacting to it [54]. In the rehabituation that can be perceived by conspecifics. It therefore paves phase, the occurrence of looking increased when negative the way for the investigation of the evolutionary calls were played compared with positive calls. In evolu- importance of emotional perception in non-human tionary terms, it is important to attend to negative signals animals [19, 25, 26]. with life-threatening consequences, and the responses of As expected, during the habituation phase, goats grad- the goats are in line with this expectation [2, 48, 55, 56]. ually reduced the duration of looking towards the sound During the habitation phase, there was a tendency for source, and heart rate also decreased, suggesting that heart-rate variability to be higher when positive calls were habituation to the valence of the stimuli occurred. Later, played back compared to negative ones. In addition, heart- the occurrence of looking increased immediately after rate variability was higher in the rehabituation phase when the second call of dishabituation, and decreased during the calls were positive. Several studies proposed that rehabituation, suggesting that goats perceived the heart-rate variability is a reliable indicator of emotional change in call valence. Since all calls played back were of valence in non-human animals [42, 43, 57, 58]and the same call type, this suggests the ability of goats to humans [59 , 60]. However, we suggest that this has to be perceive the calls as different based solely on valence further investigated, especially when the situations indu- information [47–49]. The calls played during the disha- cing positive and negative emotional states are charac- bituation phase differed in valence compared to those terised by different levels of arousal [7, 61, 62]. In the played in the habituation phase, but were not different present study, positive calls induced higher heart-rate in amplitude (loudness), because the stimuli had been variability of goats, indicating greater involvement of the rescaled to the same maximum amplitude or onset. The parasympathetic over the sympathetic system during the delay in the behavioural response, in comparison with habituation phase [39]. Thus, we suggest that goats were the faster physiological reaction, could be explained by experiencing relatively low levels of arousal throughout the way in which acoustic stimuli are processed. Stimuli the dishabituation and rehabituation phases. The HR was that are loud and have abrupt onsets are more efficient low throughout the phases of the playback and vagal at inducing responses [50]. These stimuli induce visible activation was high in response to positively-valenced calls changes within a short period of time (10 ms) at behav- because HRV increased upon hearing positive calls. ioural (e.g. stopping ongoing activity, moving the body Heart rate gradually decreased during the habituation towards the source of the noise) and physiological levels phase and did not increase when the valence of the call (increase in heart rate and blood pressure), similarly to changed, both in the dishabituation and rehabituation those that we would expect to occur when, for example, phases. Heart rate is usually affected by the type of a first call of habituation is played [50, 51]. This strong signal presented and the physiological state of the animal reaction is caused by direct circuits connecting the audi- [63]. In domestic ungulates, this parameter increases tory nerve to posterior parts of the brain (i.e. nucleus mainly when hearing sudden noises, during novel object pontis caudalis of the reticular formation; [50]). In our presentation and when unpredictable events are pre- experiment, by contrast, the calls played during the sented [64]. Goats experienced a stable level of HR dur- dishabituation phase differed from those played in the ing dishabituation and rehabituation phases, even when habituation phase only by their context of production. the call valence changed. Nevertheless, heart-rate vari- The absence of variation in the heart rate could be due to ability, suggests that goats discriminated the subtle infor- the fact that the stimuli used had a relatively similar mation about valence conveyed by the call structure. arousal level and, in addition, the call used in each se- To summarise, our combined behavioural and physio- quence belonged to the same subject. We suggest that the logical measures provide evidence that non-human subtle and slower behavioural response could be due to animals can discriminate subtle changes within call types the regulation of the emotional changes in the listener. as a result of the emotional valence experienced by the The regulation of emotional states is controlled by cholin- producers. Perceiving the emotional state of another in- ergic and dopaminergic systems [51, 52] and potentially dividual through its vocalisations and being affected by by the amygdala receiving projections from the , those vocalisations have a strong adaptive value consid- directly connected with the cochlear root neurons [53]. ering the dynamics of social organisations where, for We interpret the short time delay (2 calls over 40 s) in example, group size and composition changes over time. goat responses to the dishabituation calls as resulting from Many social animals live under environmental conditions the time needed to process the emotional change con- where individuals are not always in visual contact with one veyed in the call [51–53]. Indeed, the response observed another during the day or night [65, 66], and therefore, during the first call of dishabituation (low looking dur- could acquire an evolutionary advantage through the dis- ation) might be explained by a “freezing” response, where crimination of the emotional content of conspecifics’ calls goats were gathering more information on the valence of [67]. Furthermore, expressing emotions using vocalisations Baciadonna et al. Frontiers in Zoology (2019) 16:25 Page 6 of 11

and being able to detect and share the emotional state of a Table 1 Goats tested and experimental design. PNP indicates a conspecific may facilitate motor coordination among the Positive (habituation) - Negative (dishabituation) - Positive individuals in a group and strengthen social bonds and (rehabituation) sequence; NPN indicates a Negative (habituation) - – group cohesion [21, 22, 68–70]. Positive (dishabituation) Negative (rehabituation) sequence. FEFR indicates sequences built with FEeding anticipation and feeding Conclusion FRustration calls; FRFE indicates sequences built with feeding FRustration and FEeding anticipation calls; FEIS indicates sequences Although a substantial amount of research has been built with FEeding anticipation and ISolation calls and ISFE indicates done to investigate emotion expression in non-human sequences built with ISolation and FEeding anticipation calls animals and to reveal their importance for survival at D Sex Age Group Session Playback Sex Sequence the individual level, the investigation of emotion percep- 1 Male 8 1 1 Male PNP (FEFR) tion in group living animals is sparse [21]. Here we provide evidence for the ability of non-human animals 2 Male NPN (FRFE) to discriminate emotions conveyed in calls emitted by 2 Male NA 1 1 Male PNP (FEIS) conspecifics. We also provide evidence for the impact of 2 Male NPN (ISFE) emotionally-valenced calls on the behaviour (occurrence 3 Male 7 1 1 Male PNP (FEFR) of looking) and physiological responses (HRV) of goats. 2 Male NPN (FRFE) When the behavioural and physiological parameters are 4 Female 9 1 1 Female PNP (FEIS) combined, our results suggest that non-human animals are not only attentive, but might also be sensitive to the 2 Female NPN (ISFE) emotional states of other individuals. 5 Female 9 1 1 Female PNP (FEFR) 2 Female NPN (FRFE) Methods 6 Female 4 1 1 Female PNP (FEIS) Subjects and experimental apparatus 2 Female NPN (ISFE) The study was carried out at Buttercups Sanctuary for 7 Male 12 1 1 Female PNP (FEFR) Goats (http://www.buttercups.org.uk) in Kent, UK. At the sanctuary, goats are released into a large field during 2 Female NPN (FRFE) the day and are confined indoors either in individual or 8 Male 4 1 1 Female PNP (FEIS) shared pens (average size = 3.5 m2) at night. Goats have 2 Female NPN (ISFE) ad libitum access to hay, grass, and water and are also 9 Male 9 1 1 Female PNP (FEFR) fed with a commercial concentrate according to their 2 Female NPN (FRFE) health condition and age. In total, 24 adult goats (12 10 Female 5 1 1 Male PNP (FEIS) females and 12 castrated males) of different breeds and ages (Table 1) were tested from May to September 2015, 2 Male NPN (ISFE) at Buttercups Sanctuary for Goats in Kent (UK). An 11 Female NA 1 1 Male PNP (FEFR) experimental arena (7 m × 5 m) was set up and placed in 2 Male NPN (FRFE) one of the fields where the goats are released during the 12 Female 8 1 1 Male PNP (FEIS) day. The arena consisted of a rectangular area composed 2 Male NPN (ISFE) of a start pen connected by a gate to a central arena 13 Male 7 2 1 Male NPN (FRFE) made with a commercial opaque agricultural metal fence (Fig. 4). A loudspeaker was placed outside the perimeter 2 Male PNP (FEFR) of the arena, on the opposite side to the main gate. The 14 Male 9 2 1 Male NPN (ISFE) speaker was not visible to the goats and was concealed 2 Male PNP (FEIS) with camouflage netting. 15 Male 10 2 1 Male NPN (FRFE) 2 Male PNP (FEFR) Sound recordings 16 Female 3 2 1 Female NPN (ISFE) The vocalisations used in this study were obtained from a previous study [7] conducted at the same location. The calls 2 Female PNP (FEIS) selected belonged to goats that did not share a pen with the 17 Female 3 2 1 Female NPN (FRFE) subjects during the night, or to goats that were no longer at 2 Female PNP (FEFR) the sanctuary at the time of testing. Calls were recorded at 18 Female 11 2 1 Female NPN (ISFE) – distances of 3 5 m from the focal animal using a Sennhei- 2 Female PNP (FEIS) ser MKH-70 directional microphone (frequency response 19 Male NA 2 1 Female NPN (FRFE) 50–20,000 Hz; max SPL 124 dB at 1 kHz) connected to a Marantz PMD-660 digital recorder (sampling rate: 44.1 2 Female PNP (FEFR) Baciadonna et al. Frontiers in Zoology (2019) 16:25 Page 7 of 11

Table 1 Goats tested and experimental design. PNP indicates a and the other one was recorded while its pair mate was eat- Positive (habituation) - Negative (dishabituation) - Positive ing; 3) isolation (negative, low arousal), in which the tested (rehabituation) sequence; NPN indicates a Negative (habituation) - goats were recorded while isolated in a pen alone for 5 min – Positive (dishabituation) Negative (rehabituation) sequence. FEFR away from the other goats but within their usual daytime indicates sequences built with FEeding anticipation and feeding range, after 3 days of habituation to this situation. The FRustration calls; FRFE indicates sequences built with feeding changes in the behaviour and physiology of the subjects in FRustration and FEeding anticipation calls; FEIS indicates sequences built with FEeding anticipation and ISolation calls and ISFE indicates these three contexts were examined. The arousal and the sequences built with ISolation and FEeding anticipation calls valence of each recording context were determined using (Continued) physiological and behavioural indicators of emotions (used D Sex Age Group Session Playback Sex Sequence to validate of the emotional arousal and valence; [7]. Food anticipation and food frustration induced higher arousal 20 Male 4 2 1 Female NPN (ISFE) compared to isolation. Food anticipation and food frustra- 2 Female PNP (FEIS) tion were also associated with lower heart-rate variability, 21 Male 13 2 1 Female NPN (FRFE) higher respiration rate, more movements, more calls, more 2 Female PNP (FEFR) time spent with ears pointing forwards and less time with 22 Female NA 2 1 Male NPN (ISFE) ears on the side. In the food anticipation condition, goats 2 Male PNP (FEIS) had their ears oriented backwards less often and spent more time with their tails up compared to the food frustra- 23 Female 5 2 1 Male NPN (FRFE) tion and isolation conditions [7]. The detailed vocal param- 2 Male PNP (FEFR) eter analysis identified six acoustic parameters affected by 24 Female 12 2 1 Male NPN (ISFE) the arousal. F0 contour over time and energy quartile in- 2 Male PNP (FEIS) creased with arousal, whereas the first formant decreased. F0 variation within the call was influenced by valence and kHz with amplitude resolution of 16 bits in WAV format). decreased from negative to positive valence. The acoustic Three different contexts inducing emotions were consid- structure of the calls is described in more detail in Briefer ered: 1) food anticipation (positive, high arousal), in which et al. [7]. the goats, tested in pairs in two adjacent pens, learned to anticipate a food reward after three days of training and Playback experiments and exclusion criteria were recorded on the fourth day when the experimenter The habituation-dishabituation-rehabituation paradigm approached the tested goats with a bucket of food; 2) food (modified from Charlton et al., [37, 48, 49]) was used to in- frustration (negative, high arousal), in which only one of vestigate whether goats are able to perceive conspecific the goats in a pair received food from the experimenter, vocal expression of emotional valence. The paradigm is

Fig. 4 Experimental enclosure. The experimental apparatus (7 m × 5 m) consisted of a start pen connected by a door to a central arena. The loudspeaker was placed at the far end of the arena (outside the perimeter) and was covered with hunting net and natural vegetation. The experimenter remained inside the start pen during the tests, out of view, behind a PVC garden screening fence Baciadonna et al. Frontiers in Zoology (2019) 16:25 Page 8 of 11

based on the repeated presentation of a stimulus, for isolation (dishabituation) – food anticipation (rehabitua- example a positive call produced while a goat was experien- tion) calls, (hereafter, “FEIS”); and five sequences included cing a given emotional valence, to a subject (habituation), isolation (habituation) – food anticipation (dishabituation) followed by the presentation of a different stimulus - isolation (rehabituation) calls, (hereafter, “ISFE”). [dishabituation; in our case, calls produced while a goat was Calls within the sequence were emitted by the same experiencing a situation with emotional valence opposite to individual, but were produced in two different emotional thesituationusedduringthehabituationphase(e.g. contexts. The first nine calls (three different calls pro- negative)]. The response (behavioural and/or physiological) duced in a given context – food anticipation, food frus- of the subject should indicate whether the element that dis- tration or isolation - repeated three times each and tinguishes the two stimuli (in our case, change in valence) combined in random order) constituted the habituation is conspicuous enough to be detected. A reduction in the phase (H); the following three calls (three different calls response of the subject (habituation) after a repeated pres- produced in a context of opposite valence compared to entation of the stimulus, followed by an increment in the the habituation calls, and combined in a random order) response when a new stimulus is presented (dishabituation) constituted the dishabituation phase (D); and the final would indicate that the two stimuli are perceived as differ- call (a single call randomly selected from the habituation ent [48, 49, 71]. After the dishabituation, the stimulus used phase) constituted the rehabituation phase (R). in the habituation is presented again (rehabituation), in Each vocalisation was broadcasted from a Mackie order to ensure that the response occurring during the Thump TH-12A loudspeaker (LOUD Technologies Inc., dishabituation is robust and not a random consequence of Woodinville, WA; frequency response: 57 Hz - 20 kHz ± a renewal of attention [48, 49]. 3 dB) connected to an active box to boost the sound Twenty four sessions (six goats in total, playback se- (Active Box DI-100 Fame) and to an audio player (Tech- quences played FEFR = 5, FRFE = 7, FEIS = 8, ISFE = 4) nika MP111), at an approximately natural amplitude were excluded from the final analysis because: 1) subjects (88.99 ± 0.93 dB) measured at 1 m using an ASL-8851 did not react to the first habituation call, i.e. individuals sound level meter. The original duration of the calls was did not look towards the source of the playback during maintained, in order not to remove any information the first call of habituation, and/or 2) subjects failed to contained in their structure (feeding = 0.71 ± 0.02 s; frus- habituate, defined as sessions where the time spent look- tration = 0.70 ± 0.03 s and isolation = 0.71 ± 0.02 s). The ing towards the speaker during the last playback of the peak amplitude of each call had been equalised during habituation phase was more than two times longer than the preparation of the sequences. The presentation order the first playback of the habituation phase [37]. of the playback sequences was balanced within each group of 12 subjects (tested in the same day), so that Playback sequence and procedure half of the subjects experienced first the Positive – Each playback sequence consisted of 13 calls, separated by Negative - Positive (PNP) sequence and the opposite a time interval of 20 s. Only good quality calls with low Negative – Positive - Negative (NPN) sequence in the background noise were selected to prepare the playback following session. The other half of the group experi- sequences as follows: three calls per individual with a enced NPN first and PNP in the following session. The signal-to-noise ratio > 10 dB were selected from eight indi- sex of the goat that produced the calls used in the play- viduals in the food anticipation context, from six individ- back sequence was counterbalanced within and between uals in the food frustration context and from five subjects (half the males and half the females were tested individuals in the isolation context (i.e. 57 calls in total) with same sex playbacks and the other half with opposite within the original pool of 180 calls (i.e. 40 calls in food sex playback). Overall, each subject was tested on two anticipation; 80 calls in food frustration and 60 calls in iso- different days, with one session per day, and a three-day lation; [7]. In order to test if the valence of the calls was interval between sessions. perceived regardless of context (two contexts of negative Before the experiment started, goats were released valence; frustration and isolation) and order (i.e. which twice (i.e. one for each consecutive day) for 5 min inside valence was used for the habituation or dishabituation the arena to familiarise with the experimental setup. phase), the sequences included the following combina- During the test phase, individuals were gently brought tions of valence and context: six sequences included food to the start pen, where a familiar experimenter placed anticipation (habituation) – food frustration (dishabitua- the heart rate monitor BioHarness belt around the goats’ tion) – food anticipation (rehabituation) calls, (hereafter, thorax. When a clear electrocardiogram (ECG) was “FEFR”); six sequences included food frustration (habitu- obtained, the main gate that provided access to the ation) – food anticipation (dishabituation) – food frustra- central arena was opened. After 30 s, the first playback tion (rehabituation) calls, (hereafter, “FRFE”); five call was played and the session continued until the last sequences included food anticipation (habituation) - call was played. Baciadonna et al. Frontiers in Zoology (2019) 16:25 Page 9 of 11

Behavioural and physiological data collection and significant decrease in occurrence of looking and in HR analyses throughout the phase). Subsequently, responses to the The duration of looking towards the speaker was mea- last habituation call (H9) were compared to those of the sured and defined as the time from when the subject first dishabituation call (D10). Responses were also com- directed the head towards the playback location (start) pared to dishabituation calls D10 vs D11, and D11 vs until when the head was turned away and the animal D12, to investigate the response pattern within the dish- stopped looking (end), within the 20 s following each abituation phase. Finally, responses to the dishabituation call. If the subjects were already looking towards the calls (D10, D11, and D12) were compared to those of speaker when one of the calls of a sequence was broad- the rehabituation call (R13). The model selection and casted, then this behaviour was considered to begin at the variable considered were call number (1 to 13; or a the onset of the playback [48]. When the goat looked combination of these for further post-hoc tests) and call away and then looked back to the speaker within the 20 valence (positive or negative), as well as their interaction s following each call, the time was scored again. The as fixed effects. The duration of the measurement period total duration of looking towards the sound source was (9.34 ± 0.17 s) was also included as a control factor in calculated for each subject and for each of the 13 calls. the model carried out on RMSSD, because it could All trials were video recorded using a digital video cam- potentially affect this value. The factor “Session” [1 and era placed at the entrance of the arena (Sony HDR- 2] nested within the identity of the goats (“ID”) nested CX190E). The videos were analysed frame by frame within “Group” [1 and 2] was included as a random fac- using QuickTime player (Apple Inc.). A second observer, tor, crossed with the identity and the sex of the goat pro- blind to the experimental hypothesis, scored 30% of the ducing the playback calls. Non-significant interactions sessions to test the reliability of the parameters mea- between call number and valence were removed from sured by the two observers. Inter-observer agreement the models [74]. The statistical significance of the factors for the behaviour scored was high (Spearman rank was assessed by comparing the models with and without correlation; rs = 0.990, p < 0.001). the factor included using a likelihood-ratio test. When The physiological parameters were recorded using a an interaction effect was found, further post-hoc com- non-invasive Bluetooth device (EC38 Type 3, BioHarness parisons were performed using a Tukey HSD test. Physiology Monitoring System, Zephyr Technology Q–Q plots and scatterplots of the residuals of the Corporation, Annapolis, MD, USA) fixed to a belt placed model were checked visually for normal distribution and around the goat’s chest. A small patch of hair (7 cm X homoscedasticity. In order to meet the model assump- 15 cm) was clipped before the experiment in order to tions, HR was log-transformed. HR (log-transformed) obtain a clearer ECG trace. This procedure took place a and RMSSD were input into LMMs fit with Gaussian week before the testing to avoid any confounding effects family distribution and identity link function. The occur- of being manipulated. The continuous ECG trace was rence of looking towards the speaker did not meet the transmitted in real time to a laptop (ASUS S200E) and assumptions despite log-transformation. It was thus registered using the software AcqKnowledge v.4.4 (BIO- transformed to binary data (looked at the speaker = 1; PAC System Inc.). During the playbacks, we entered did not look = 0) and input into a GLMM fit with bino- visible markers in the ECG trace at the beginning of mial family distribution and logit link function. each call to be able to link the physiological data to the Acknowledgements specific calls and phases of the experiments. The time of We are grateful to Ednei dos Santos, Katherine Herborn, Christian Nawroth, occurrence of each heart beat identified on the ECG Monica Padilla de la Torre, Stuart Semple, Caroline Spence, and two trace was extracted during the 20 s following each call. anonymous reviewers for their helpful comments on the manuscript. We thank the staff members and the volunteers at Buttercups Sanctuary for HR and HRV (measured as root mean square of succes- Goats (www.buttercups.org.uk) for their excellent help and for providing free sive inter-beat interval differences, RMSSD) were further access to the animals. calculated from the extracted heart beats on the longest selection possible within 20 s. Authors’ contributions LB, EFB, LF and AGM conceived/designed the study; EFB collected the recordings; LB conducted experiments; LB and EFB analysed the data; LB, EFB, LF and AGM Data analysis wrote the manuscript. All authors gave final approval for publication. Analyses were conducted using Linear and Generalised Funding Mixed-Effects Models (lmer function, lme4 library; During this study, Livio Favaro was supported by the University of Torino Pinheiro 2000) in R v.3.2.2 [72, 73]. First, the occurrence through a System S.p.A. research grant for bioacoustics, and Elodie F Briefer of looking towards the speaker, HR and RMSSD were by a Swiss National Science Foundation fellowship (PZ00P3_148200). compared over the nine calls played during the habitu- Availability of data and materials ation phase (H1-H9) to determine whether goats habitu- All data generated or analysed during the study are included in this ated to the sounds throughout this phase (indicated by a submitted article and its supplementary information file. Baciadonna et al. Frontiers in Zoology (2019) 16:25 Page 10 of 11

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